Branching morphogenesis of the ureteric bud (UB) to generate the renal collecting duct system is a critical process for the formation of a normal urinary tract and kidney. Abnormalities in this process cause birth defects such as renal agenesis or hypodysplasia, low nephron number, or congenital obstructive uropathies. Low nephron number may promote the progression of renal diseases and the development of hypertension. Thus, a better understanding of the genetic controls and cellular events underlying UB branching could lead to new strategies to prevent such birth defects, repair renal damage, or grow artificial kidneys. While many of the genes required for normal UB branching morphogenesis have been identified, ultimately it is the specific cell behaviors controlled by these genes that cause correctly patterned epithelial growth and branching. These cellular behaviors are, in general, poorly understood. This project focuses on the role of GDNF signaling through the Ret receptor, a signaling event that is critical for kidney development in mice and humans. Our recent studies show that the branching UB epithelium undergoes extensive cell rearrangements, many of which are controlled by Ret. We hypothesize that these cell rearrangements are a major force driving normal UB branching. We seek to describe the nature of these cell movements, their control, and their importance for kidney development, using a variety of state-of-the art genetic and imaging technologies. We propose to use several genetic methods to label individual, wild-type ureteric bud cells with fluorescent proteins, or to generate labeled clones of mutant UB cells in developing kidneys. We then follow their behaviors during renal development in culture, via high-resolution, 4-dimensional (4D) time-lapse imaging. In Aim 1, we investigate the importance of a new type of cell motility, linked to mitosis, which occurs in the UB tip epithelium. We ask if these mitosis-associated cell movements are non-random in direction, if they influence the developmental fate of the daughter cells, and if they are disrupte by mutations in several candidate genes. In Aim 2 we perform global 4D tracking of every nucleus in a branching UB tip, thus analyzing in unprece- dented detail the variety of cell movements that occur during normal, as well as abnormal, UB branching. We also investigate the extensive protrusive activity in UB tip cells, observed in high-resolution imaging studies, and examine its role in epithelial cell motility. In Aim 3, we use several powerful methods of clonal genetic analysis to continue our investigation of how Ret signaling, as well as the activity of several genes acting upstream or downstream of Ret, influences cell movements during UB branching. Overall, the proposed research should advance the field by more deeply elucidating the role of GDNF/Ret signaling in UB cell behaviors; by providing a thorough picture of cell motility in the UB epithelium, its regulation, and its contribu- tion to normal and abnormal branching morphogenesis; and by providing new tools, methods and paradigms for studying how other genes and signaling pathways affect the behaviors of renal epithelial cells in vivo.